Carotenoids are pigments that are ubiquitous throughout nature and synthesized by all photosynthetic organisms, and in some heterotrophic growing bacteria and fungi. Carotenoids provide color for flowers, vegetables, insects, fish and birds. Colors of carotenoid range from yellow to red with variations of brown and purple. As precursors of vitamin A, carotenoids are fundamental components in our diet and they play additional important role in human health. Because animals are unable to synthesize carotenoid de novo, they must obtain them by dietary means. Thus, manipulation of carotenoid production and composition in plants or bacteria can provide new or improved source for carotenoids. Industrial uses of carotenoids include pharmaceuticals, food supplements, animal feed additives, and colorants in cosmetics, to mention a few.
Industrially, only a few carotenoids are used for food colors, animal feeds, pharmaceuticals, and cosmetics, despite the existence of more than 600 different carotenoids identified in nature. This is largely due to difficulties in production. Presently, most of the carotenoids used for industrial purposes are produced by chemical synthesis; however, these compounds are very difficult to make chemically (Nelis and Leenheer, Appl. Bacteriol. 70:181–191 (1991)). Natural carotenoids can either be obtained by extraction of plant material or by microbial synthesis. However, only a few plants are widely used for commercial carotenoid production and the productivity of carotenoid synthesis in these plants is relatively low. As a result, carotenoids produced from these plants are very expensive.
One way to increase the productive capacity of carotenoid biosynthesis is to apply recombinant DNA technology (reviewed in Misawa and Shimada, J. Biotech., 59:169–181 (1998)). It is desirable to produce carotenoids in non-carotenogenic bacteria and yeasts, thereby permitting control over quality, quantity and selection of the most suitable and efficient producer organisms. The latter is especially important for commercial production economics (and therefore availability) to consumers.
CrtZ-type carotenoid hydroxylases are a class of enzymes that introduce hydroxyl groups to the β-ionone ring of the cyclic carotenoids, such as β-carotene or canthaxanthin, to produce hydroxylated carotenoids. Examples of such carotenoids include astaxanthin, β-cryptoxanthin, zeaxanthin, 3-hydroxyechinenone, 3′-hydroxyechinenone, adonirubin, adonixanthin, tetrahydroxy-β,β′-caroten-4,4′-dione, tetrahydroxy-β,β′-caroten-4-one, caloxanthin, erythroxanthin, nostoxanthin, flexixanthin, 3-hydroxy-γ-carotene, 3-hydroxy-4-keto-γ-carotene, bacteriorubixanthin, bacteriorubixanthinal, and lutein.
Carotenoid hydroxylase genes have been reported from a variety of bacterial, fungal, algal, and plant species. Examples of several species include, but are not limited to Pantoea stewartii (WO 03/016503; WO 02/079395), Erwinia uredovora (EP 393690 B1; Misawa et al., J. Bacteriol., 172(12):6704–6712 (1990)), Erwinia herbicola (Hundle et al., Mol. Gen Genet., 245(4):406–416 (1994); Hundle et al., FEBS Lett., 315(3):329–334 (1993); Schnurr et al., FEMS Microbiol. Lett., 78(2–3):157–161 (1991); and U.S. Pat. No. 5,684,238), Agrobacterium aurantiacum (Misawa et al., J Bacteriol., 177(22):6575–6584 (1995); U.S. Pat. No. 5,811,273), Alcaligenes sp. (U.S. Pat. No. 5,811,273), Flavobacterium sp. (U.S. Pat. No. 6,677,134; U.S. Pat. No. 6,291,204; U.S. 2002147371; WO 2004029275; and Pasamontes et al., Gene, 185(1):35–41 (1997)), Paracoccus sp. (CN 1380415), Haematococcus pluvialis (WO 00/061764; Linden, H., Biochimica et Biophysica Acta, 1446(3):203–212 (1999)), and plant species such as Arabidopsis thaliana (Tian, L. and DellaPenna, D., Plant Mol. Biol., 47(3):379–388 (2001); U.S. 2002102631). Many of these β-carotene hydroxylase genes have been recombinantly expressed in microbial host cells. However, there is a need to identify additional novel crtZ hydroxylase genes useful for genetically engineering commercially-suitable microorganisms for the production of hydroxylated carotenoids, such as astaxanthin and zeaxanthin.
Additionally, there is a particularly important need to identify CrtZ hydroxylases having relatively low to moderate nucleic acid sequence identity (i.e. <70% nucleotide sequence identity) for stable expression of multiple hydroxylase genes as highly homologous genes would be difficult to integrate and tend to result in genetic instability (i.e. undesirable homologous recombination). CrtZ genes having a divergent nucleotide sequences (relative to one another) are most suitable for expressing multiple hydroxylases in a single recombinant host cell. This is especially important when hydroxylase activity becomes the rate-limiting step in the carotenoid biosynthesis pathway. Increasing the number of crtZ genes that can be simultaneously expressed in the production host is expected to increase carotenoid production, assuming that the pool of available substrates is not limiting. This is particularly important when optimizing recombinant production of the desired product (i.e. carotenoids).
The problem to be solved therefore is to provide novel crtZ hydroxylase genes useful for engineering production of carotenoids (i.e. zeaxanthin and astaxanthin).